The ability of antigen presenting cells (APC) to process proteins to antigenic peptides has been well documented, and much is now known about the structures of the major histocompatibility complex (MHC) proteins as well as the T cell receptor which recognizes antigen complexed with the MHC proteins. In contrast, the sequence of events which leads from initial recognition of a non-antigenic protein by an APC to presentation of peptide fragments as antigens is less well understood. This proposal describes experiments designed to investigate the intracellular events which take place when proteins are internalized by APC. The ultimate goal of these experiments is to define the criteria that govern the conversion of circulating proteins to peptides recognized as antigenic by helper T cells. Evidence is now available that processing of proteins that are destined to become antigenic peptides occurs in an intracellular, acidic compartment, possibly an endosome. Recently, an acidic protease, cathepsin D, was identified in the endosomes of alveolar macrophages, however, the ability of these cells to present antigen as a result of the action of this protease has not been determined. To evaluate the role of endosomal proteolysis in antigen presentation, experiments will be carried out to answer the following questions: First, where inside an APC does the internalized protein first encounter proteolytic activity? Second, where inside an APC do MHC class II molecules first encounter the peptide products of protein cleavage? Third, how do antigenic peptides and MHC class II molecules recycle to the plasma membrane of an APC, prior to recognition by T cell receptors? Fourth, is the possession of endosomal proteolytic activity common to APC other than macrophages, for example, B cells and dendritic cells? The fate of the protein after arrival in endosomes will be determined to find out whether it is processed by any proteolytic activity, whether it is delivered to lysosomes, or recycled to the plasma membrane. The fate of each peptide fragment and its association with MHC class II molecules will be determined.